ZooGene Sample Preservation Protocol

Unsorted zooplankton samples can be preserved in 95% ethyl alcohol
for later molecular genetic analysis. Samples intended for use
in the molecular characterization of a particular species (i.e., molecular
systematics) should contain 10 - 20 adult individuals (ideally including
both males and females) of the targeted species. Samples intended to
allow examination of geographic variation within a species (i.e.,
population genetics) should include 30 - 50 individuals (ideally adult
females) per sample.

In most cases, the sender should confirm the identification of the desired
species. The selected individuals should be removed from the sample and
place in a small glass vial (5 - 20 ml) with a plastic top that prevents
evaporation. Please include a label inside the vial, writing in pencil.

Please follow the sample preservation protocol below.

ALCOHOL PRESERVATION of samples for molecular analysis

Samples collected with minimum damage to plankton are preferred
(i.e., short tows, rapidly preserved). Suitable net mesh sizes can vary
between 100 um to 333 um for copepods, 550 um for euphausiids.
Samples need not be quantitative; non-quantitative aliquots or splits
of samples are also fine.

Because of the rapid rate of DNA destruction, zooplankton samples should
be preserved IMMEDIATELY following collection, while the
individuals appear lively, transparent, and healthy. Individuals that
appear moribund or dead should not be preserved for molecular analysis.

Drain samples of excess seawater (over nitex mesh of same size or
smaller than the net used for collection).

Place sample in round-sided, flat-bottomed glass jar (i.e., avoid
jars with corners where putrefaction can occur and opaque jars where
that sample cannot be easily observed). Add 95% ethyl alcohol; there must
be 3 to 4 times more alcohol than plankton volume - especially for larger
individuals, such as euphausiids. We recommend removing fish, gelatinous
plankton, and other large non-essential organisms from the samples to be
preserved in alcohol.

NOTE: DENATURED ALCOHOL CANNOT BE USED FOR SAMPLE PRESERVATION FOR
MOLECULAR ANALYSIS. 100% ETHYL ALCOHOL SHOULD ALSO NOT BE USED FOR
PRESERVATION FOR MOLECULAR ANALYSIS: it frequently retains traces of the
benzene, or other solvent, used to displace the remaining water from the
alcohol.

Place a label inside the jar or vial, writing in pencil. Unprinted labels
are preferred since the ink may dissolve in the alcohol. Use small labels
made from acid-free paper. (We have discovered that some labels
change the sample pH significantly, especially in small volumes).

After 24 hours, drain off the alcohol (using Nitex or mesh of the same
size as the collection net or smaller) and replace with fresh
alcohol. Check periodically for evaporation and putrefaction. Continue
changing the alcohol daily until the liquid remains clear (pigment
leaching is OK, but flocculent matter is not).

AFTER PRESERVATION

The samples can be mailed in small packages via airmail. The vials should
be wrapped in parafilm or other material to prevent leakage. Placing
vials in styrofoam molds for support is ideal; they should be well
wrapped to prevent breakage. We usually label the contents as "scientific
samples" and claim "no commercial value" for customs purposes. Samples
may be sent collect or cash-on-delivery (COD) to Ann Bucklin at the
University of New Hampshire.